Microbial Fermentation of Polyethylene Terephthalate (PET) Plastic Waste for the Production of Chemicals or Electricity

Abstract Ideonella sakaiensis (I. sakaiensis) can grow on polyethylene terephthalate (PET) as the major carbon and energy source. Previous work has shown that PET conversion in the presence of oxygen released carbon dioxide and water while yielding adenosine triphosphate (ATP) through oxidative phosphorylation. This study demonstrates that I. sakaiensis is a facultative anaerobe that ferments PET to the feedstock chemicals acetate and ethanol in the absence of oxygen. In addition to PET, the pure monomer ethylene glycol (EG), the intermediate product ethanol, and the carbohydrate fermentation test substance maltose can also serve as fermenting substrates. Co‐culturing of I. sakaiensis with the electrogenic and acetate‐consuming Geobacter sulfurreducens produced electricity from PET or EG. This newly identified plastic fermentation process by I. sakaiensis provides thus a novel biosynthetic route to produce high‐value chemicals or electricity from plastic waste streams.

Nuclear magnetic resonance (NMR) spectroscopy. 1 H and 13 C NMR spectra were recorded on a Bruker Neo Prodigy 400 MHz spectrometer (equipped with a prodigy BBO cryoprobe) in a mixture of H2O-D2O (95-5%) and a Bruker Avance III 500 MHz spectrometer (equipped with a BBO smart probe) in a mixture of H2O-D2O (95-5%) using H2O suppression by pre-saturation at room temperature. All samples were filtered with syringe filters (Millex-GP, pore size 0.22 µm, Merck) prior to analysis. 570 µL of the filtrate was added to 30 µL of 5 mM TSP in D2O to give 600 µL sample for NMR analysis. Chemical shifts are referenced relative to the protium / 1 H signal of TSP: δ = 0.00 ppm and TSP was used as internal standard for the quantification of all liquid products. Quantification was executed with TopSpin 4.0.8. The representation of the NMR spectra was done using MestReNova 12.0.0 and the spectra were corrected using automated phase and baseline correction and additional manual baseline correction for the residual H2O peak in spectra recorded without H2O suppression.
Scanning electron microscopy (SEM). SEM was carried out on a TESCAN MIRA3 FEG-SEM. All samples were fixated by incubation in glutaraldehyde solution (2.5% in H2O) for 30 min. After washing in bicarbonate-buffered medium the dried samples were further fixated by incubation in osmium tetroxide for a minimum of 4 h. Then the samples were dehydrated by incubation in a series of ethanol solutions with increasing ethanol concentration (50, 70, 90, 100%) for 30 min each. After drying, all samples were additionally sputtered with a 10 µm layer of Pt prior to the SEM analysis.
Culturing and handling of bacteria. I. sakaiensis (NBRC 110686) was purchased from NBRC Japan and cultured in 15 mL NBRC no. 802 broth (Table S1) overnight under aerobic conditions. G. sulfurreducens PCA (ATCC 51573) was purchased from DSMZ Germany and cultured in 15 mL bicarbonate-buffered medium (Table S3) for 3 days under anaerobic conditions. Sodium acetate (20 mM) and sodium fumarate (50 mM) were added to serve as the electron donor and acceptor, respectively during G. sulfurreducens growth. Anaerobic vials and electrochemical cells were always prepared by purging 15 mL bicarbonate-buffered medium with a gas mixture of N2-CO2 (80-20%) for 1 h before and 15 min after the inoculation. All inoculated serum vials (aerobic and anaerobic) were kept in a shaking incubator (INCU-Shake MIDI, SciQuip) at 30 °C and 300 rpm for the above specified times. The concentration of all bacteria suspensions after the growth period was determined by measuring the optical density at 600 nm (OD600) using a UV-vis spectrometer (Varian Cary 50, Agilent Technologies). As-grown cells were centrifuged (Centrifuge 5804, Eppendorf) for 4 min at 7000 rpm and then resuspended in bicarbonate-buffered medium. This washing process was repeated three times and the resulting bacteria suspension was added to the anaerobic serum vials to obtain a final OD600 = 1. Fermentation by I. sakaiensis. Anaerobic serum vials were prepared as described above containing bicarbonate-buffered medium (15 mL) under a N2-CO2 (80-20%) atmosphere and either maltose (40 mM), PET film (60 mg), or EG (25 mM) was added as the fermenting substrate. After inoculation with I. sakaiensis the vials were kept in a shaking incubator at 30 °C and 300 rpm for up to one month. Aliquots (800 µL) were removed periodically using a syringe and prepared for NMR analysis as described above. After the experiment, the remaining PET films were washed with 70% ethanol and water and dried at 40 °C overnight before post-experiment quantification. Isotopic experiments were performed in the presence of 13 C labelled EG or ethanol. For experiments with killed bacteria (Table S4), I. sakaiensis was treated in an autoclave (Prestige Medical, Portable Autoclave Classic Media, 121 °C) before addition to the anaerobic serum vials. All experiments were performed in triplicate.
Co-culturing of I. sakaiensis and G. sulfurreducens in an electrochemical cell. Inverse opal-indium tin oxide (IO-ITO) electrodes (geometrical surface area: 0.25 cm 2 , thickness: 40-45 µm, macropore size: 8-10 µm) were prepared by a previously published coassembly method using ITO nanoparticles (< 50 nm particle size) and 10.0 µm polystyrene beads and annealing at 500 °C for 20 min with a heating rate of 1 °C min −1 (1). Co-culturing of G. sulfurreducens and I. sakaiensis was conducted in an anaerobic electrochemical cell with a three-electrode system consisting of an IO-ITO working electrode, a platinum mesh counter electrode, and a Ag/AgCl (in 3 M NaCl solution, + 0.20 V vs. SHE) reference electrode.
In the first step, a G. sulfurreducens biofilm was grown on the IO-ITO electrode following previously published procedures (1,2). In short, G. sulfurreducens (OD600 = 0.6) was added to an anaerobic electrochemical cell with bicarbonate-buffered medium (15 mL) and sodium acetate (20 mM) as the sole electron donor at a pH of 7 under a N2-CO2 (80-20%) atmosphere at 30 °C, and stirring at 400 rpm. The working electrode was poised at 0.10 V vs. SHE with a potentiostat (MultiEmStat3+). After obtaining a stable current (Figure S11), the medium was replenished by a fresh bicarbonate-buffered medium (15 mL, without acetate and planktonic G. sulfurreducens). In the second step, as-grown I. sakaiensis (OD600 = 1.2-1.4) was added as the co-culture together with either PET (60 mg) or EG (25 mM) as the sole electron donor. The co-culture was kept for several days at a pH of 7 under a N2-CO2 (80-20%) atmosphere at 30 °C, stirring at 400 rpm, and the working electrode was again poised at a potential of 0.1 V vs. SHE for several days. Aliquots (800 µL) were removed periodically using a syringe and prepared for NMR analysis as described above. Remaining PET films were quantified after the experiment as described above.          . Key proteins involved in the maltose fermentation. Maltose was initially diffused through maltoporin (LamB) and taken up by the ABC transporters, substrate binding periplasmic protein MalE, permease proteins MalF and MalG, and ATP bindin g proteins MalK. Incoming maltose was metabolized to glucose and maltodextrin by a cytoplasmic enzyme, amylomaltase (MalQ). The glucose and maltodextrin were converted into glucose-6-P and glucose-1-P by glucose kinase (GK) and maltodextrin glucosidase (MalZ), respectively. Glucose-1-P was further converted to glucose-6-P by the action of phosphoglucomutase (PGM). Glucose-6phosphate isomerase (GPI) converted glucose-6-P to fructose-6-P which further turned to fructose-1,6 biphosphate by phosphofructokinase (PFK). Fructose-1,6 biphosphate was degraded to phosphoenolpyruvate by phosphoenolpyruvate synthase (PEP) which was converted to pyruvate by pyruvate kinase (PK). Pyruvate was converted to acetyl-CoA by pyruvate dehydrogenase (PDH). In the anaerobic pathway, acetyl-CoA was converted to lactic and formic acids by the action of lactate dehydrogenase (LDH) and pyruvate formate lyase (PFL), respectively. Acetyl-CoA was also converted to ethanol by alcohol dehydrogenase (ADH) and to acetate by the combined actions of phosphate acetyltransferase (PTA) and acetate kinase (ACK). In the aerobic pathway, acetyl-CoA enters the tricarboxylic acid cycle (TCA) and the electron transport chain to yield the end products CO2 and H2O (3). All mentioned enzymes are present in the genome of I. sakaiensis (Table S2) with the exception of PFL, which has yet to be identified (4).      Tables   Table S1. Components of NBRC no. 802 broth for aerobic culturing of I. sakaiensis.